Porfiromycin antibiotic and production thereof



Nov. 23, 1965 N. BOHONOS ET AL PORFIROMYCIN ANTIBIOTIC AND PRODUCTION THEREOF FREQUENCY /A/ c/w Filed May 3, 1961 ATTORNEY United States Patent O PORFIROMYCIN ANTIBITIC AND PRODUCTION THEREF Nestor Bohonos, Nanuet, and Murray Dann, Pearl River,

N.Y., Werner Karl Hausmann, Woodcliif Lake, NJ.,

and Vladimir Zbinovsky, Nanuet, and Edward .lames Backus, Pearl River, N.Y., assignors to American Cyanamid Company, New York, N.Y., a corporation of Maine Filed May 3, 1961, Ser. N 107,572 4 Claims. (Cl. 167-65) This application is a continuation-impart of our copending application Serial No. 840,001, led September 15, 1959, now abandoned.

This invent-ion relates to a new antibiotic and to its production by fermentation, to methods for its recovery and concentration from crude solutions, and to processes for its purification.

The present invention includes within its scope the antibiotic in dilute forms, as crude concentrates, and in pure crystalline forms. These novel products are active against a variety of microorganisms including gram-positive and gram-negative bacteria. The effects of the new antibiotic on speciiic microorganisms together with the chemical and physical properties of the antibiotic differentiate it from previously described antibiotics.

The new antibiotic called porromycin (formerly designated 72) is formed during the cultivation under controlled conditions by cetrain new strains of the microcommonly referred to as the Whorled forms because of the manner in which the `sporiferous branches arise in verticillate fashion at nodes along the aerial hyp'hae. This character together with the olive-buff color of the TABLE 1 Slreptomyces verzcz'llatus (AB-929) 3,219,530 Patented Nov. 23, 1965 spores en masse dictate the placing of this organism in the Olive-Buti series of the Biverticill-us sect-ion, according to the division of the genus into sections and series as set forth by Pridham et al. in Applied Microbiology 6:52-79, 1958.

For simplicity .in the specication the new strains will be referred to as AB-929, AA-849 and AB-286. Viable cultures of these strains of Streptomyces vertcllatus have been deposited with the American Type Culture Collection, Washington, D.C. Where they have been assigned ATCC accession numbers 13,495, 13,538 and 13,539, respectively. The following is a general description of the microorganisrn Streptomyces vertcillatils strain A13-929 based on the diagnostic characteristics observed. The italicized descriptive colors are those of Ridgway-Color Standards and Color Nomenclature.

Amount of growth-Growth moderate to good on most media, spreading broadly on cornsteep liquor agar; poor growth on Czapeks, Czapeks-mannitol and nutrient agars.

Aerial mycelum and/0r en masse spore COIor.-Spores en masse Deep Olive-Bu. Aerial mycelium whitish, sometimes tinted grayish from traces of sporulation.

Soluble pigments.-None.

Reverse colon-In shades of light brown on most media, becoming darker on nutrient, Bennetts, Sabourauds and Cobalt-Amidex agars.

Morphology-Aerial mycelium velvety with vegetative filaments simple and sparingly branched. Sporophores arising as biverticillate whorls of spore chains on the straight aerial filament-s. Spores smooth walled, cylindrical, 0.6-0.8;L X 1.9-2.5@ adhering in long chains, and breaking apart with difficulty.

Temperature relatons.-Optimal range for growth and sporulation 28-37 C.

The following table shows the cultural characteristics of AB-929 when grown on several differential agar media in petri dishes for 14 days at 28 C.:

rice

Medium Amount of Growth Aerial Mycelium and Soluble Reverse Color Remarks Spore Color Pigment Waksmans Starch Agar- Moderate; somewhat Aerial mycelium whitish; None Colorless to Deep Good hydrolysis.

restricted. en masse sporulation Olive-Buti.

moderate, Deep Olive- Bull. Asparagine dextrose agar- Moderate; spreading. White aerial mycelium, ..-do Clay Color Limited colorless smdani.

lrace of grayish sporulaion. Benedict's Agar Moderate; Somewhat Aerial mycelm WhB; T10 dO do Abundant colorless t0 restricted, sporulation. yellowish exudate. Synthetic Agar (Czapeks Thin; restricted do Cream to white solution Agar). Emersons Agar Good; spreading Aerial mycelium white; no .--do Clay Color Limited colorless exudate.

sporulationxlight grayish. Nutrient Agar Light growth Aerial nycelium Whltish; ..-do Natal Brown Sectorlng prominently sporulation light, grayish. Calcium malate Agar Moderate; somewhat Aerial myceliuni white do Yellowish to Clay No clearing of malate.

restricted. tinted with grayish from Color.

light sporulation. Glucose Agar Good, spreading. White aerial myceliuin; do Clay Color 1trace oi grayisli sporulaion. Krainskys dextrose agar" Moderate. WliitiSh aerial inycelillm; do do Limited colorless to yelsporulationlight; Light lowish exudate; thin, Grayish Olive to Deep ieathery margin. O live-Bul. Potato dextrose agar do White aerial mycelium; ..-do do trace oi grayisli sporulaon. Bonnett's agar -do White aerial inyeelium; ---do Natal Brown- Moderate colorless to yeltrace of grayish to Deep lowish exudate; thin, Olive-Buti sporulation. feathery margin. Corn Steep Liquor Agni- Good. spreading Colony surface character- ---do Colorless to Deep Sectoring prominently.

broadly. ized by heavy sporulation Olive-Buff.

in Deep Olive-Buff shades. Sabourauds Msltose Agar- Moderate; somewhat White aerial mycelium; no .--do Chestnut-Brown restricted. sporulation. Cobalt-Arnidex (Hickey & Good, spreading White aerial mycelium, do Blackish Brown Moderate colorless to yellow- Tresner) Agar. tmte Wil-h grayish from ish exudate; thin, feathery the light sporulation. margin.

TABLE l-Continued Medium Amount of Growth Aerial Mycelium and Soluble Reverse Color Remarks Spore Color Pigment Yeast-Malt Agar (Yeast Moderate Aerial mycelium whit b' -..do Clay Color Colonies wrinkled.

ExtractAgar). no sporulation. Cipeks-Dox Mannitol Sparse, thin White; no sporulation -..do Colorless to wh1te gar. Inorganic Salts-Starch Moderate; Restricted Aerial myceliuni Pale -..do Warm Bui Limited colorless exudate.

Agar. Olive-Buti to whitish, Colonies raised and becoming Deep Oliveirregular. Bui in sporulating areas. sporulation moderate. Carvajals Oatmeal Agar-. Good; Spreading do do Cinnamon-Brown-- Coloniiles sectoring prominen y. Tomato paste Oatmeal do Aerial mycelium White to .-.do do Colonies elevated and fur- Agar. Pale Olive-Bul. Very rowed.

light sporulation. Tomato paste Agar Moderate Aerial growth whitish to -..do Orange-Cinnamon- Very light colorless to Pale Olive-Buti. Very yellowish exudate. light sporulition.

Some further observation of miscellaneous physiologi- 2() AB-929. If S. verticz'llatus and S. olivovertcillatus were cal tests are s-ummarized in the following table (14 days growth at 28 C. except where otherwise noted):

synonymous, the latter name would be illegitimate and the former binomial would have priority, because accord- TABLE 2 Medium Amount of Growth Aerial Mycelium and Soluble Reverse Color Remarks Spore Color Pigment Peptone-Iron 2 Agar Good. B2S positive. GeTlatiii-Agar (Tyrosinase -..do Tyrosinase positive.

es Gelatin dn Partial liquefaction. Potato Plugs Heavy Aerial mycelium white, Slight tinted grayish from trace graying of sporulation. of

. potato. Carrot Plug Good White aerial mycelium; no None sporulation. Litmus Milk Moderate White aerial growth; no Slight tan No coagulation; partial sporulation. color in peptonization; pH 6.6.

upper medium. Cellulose 2 (filter paper in Slight Growth No cellulose decomposition.

Czapeks Solution).

1 24 hours incubation. 2 21 days incubation.

There are four species in the olive buff series of Pridham et al. referred to above. These are differentiated from AB-929 as follows.

The first, S. albretl'culi, differs from AB-929 in having spore masses in shades of Pale Ochraceous-Salmom to Warm Bu, and sporulation occurs readily on many inedia. Reference cultures of S. albiretculi show cultural deviations from AB929, such as growth habit, reverse coloration, etc.

Streptomyces caespitosus is described as having aerial mycelium in yellowish lto greenish shades on several media; also, on synthetic agar colony centers are elevated and caespitose, making this species distinguishable from AB-929. S. Caesptgsus is further distinguishable from AB-929 by the smaller size of its spores, its lack of tyrosinase production, VVits* greater capacity to liquefy gelatin and by its abundant production of soluble yellowbrown pigments on many media.

Slreptomyces eurocidcus differs from AB-929 in its yellowish to brownish growth on several media, and in its lack of proteolytic action in gelatin and its failure to peptonize milk.

Neither a description nor a reference culture of Streptomyces olvoverticz'llatus is available for comparison; however, synonymy of AB-929 with this organism can be ruled out for the following reasons. When Al-929 is compared with a published description of Streptomyces vertz'cllatus (placed in the undifferentiated series of section Biverticillus by Pridham et al.), close correspondence is found in their diagnostic features. This would preclude the possibility of synonymy of our isolate with S. olivovertcillatus, since the latter would have to differ signicantly from S. vertz'cllatus in order to be a valid species, and thereby would of necessity, be different from ing to the International Code of Nomenclature of Bacteria and Viruses, the oldest legitimate name takes preference. In the present instance, S. vertcillatus was erected in 1938, While S. olvo'verticllatus was named in 1956.

It is to be understood that for the production of the antibiotic of this invention the present invention is not limited to the use of the cultures AB-929, AA-849 and AB-286 or to organisms fully answering the above growth and microscopic characteristics which are given for illustrative purposes. In fact, it is desired and intended to include the use of mutants produced from the described organisms by various means such as X-radiation, ultraviolet radition, nitrogen mustard, phage exposure and the like.

The fermentation process The process by which the new antibiotic is produced is preferably an aerobic fermentation of an aqueous nutrient medium inoculated with the new strains described above. The constituents of the fermentation medium and the conditions of the fermentation are generally those of other fermentation processes in which fungi are employed to produce antibiotics.

The nutrient medium contains an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc., an assimilable source of nitrogen such as corn steep liquor and inorganic cations such as potassium, sodium, calcium, etc., and anions like sulfate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc. are supplied as needed in the form of impurities by the other constituents -of the medium. Aeration in tanks and bottles is provided by forcing sterile air through or -onto the surface of the fermenting medium.

Shaker flask fermentation The culture AB-929 is carried at 28 C. on a yeast- Inalt agar slant of the following composition:

G./l. Yeast extract 4 Malt extract Glucose 4 Agar The pH is adjusted to 7.0 with KOH. The transfers are made directly from the slants to the inoculum medium of the following composition:

G./l. Soybean meal 20 Corn steep liquor 5 Glucose 20 CaCOa 3 One hundred milliliters of the above medium are transferred to a 500 milliliter Erlenrneyer flask, sterilized, inoculated with spore-mycelia transfer from a week old agar slant, and shaken for 48 hours at 28 C. on a reciprocating shaker (2 inch stroke, 104 cycles per minute). The final mash is used to inoculate the shaker flask fermentation medium which has the following composition:

G./l. Corn steep liquor 12.5 Corn starch 10.0 (NH4)2SO 1 5.0 Citric acid 2.0 (NHQZHPOJ, 2.0 NaCl 2.0 KH2PO4 1.5 K2HPO4 0.5 MgSO4-7H2O 0.25

The pH is adjusted to 7.0 with NaOH. Fifty milliliters of this medium are transferred to a 250 milliliter Erlenmeyer flask, sterilized, inoculated and shaken four days at 28 C. on a rotary shaker at 240 r.p.m. Samples are periodically taken, centrifuged and stored at 4 C. until assayed by the cup-plate technique. Inhibition zones of the following diameters have been obtained: 24.2 mm. for a 1:256 dilution on B. subtilis, pH 6, 20.5 mm. for a 1:64 dilution on Strep. pyogenes haemolyticus and 21.3 mm. for a 1:128 dilution on Mycobacterium No. 607.

T ank fermentation The inoculum is prepared in the following manner. An agar slant of the culture is incubated for a week. At this time, the spores and mycelium are transferred to two 500 milliliter asksA containing 100 milliliters of the inoculum medium described earlier. The flasks are then shaken on a reciprocating shaker for 48 hours. The contents are transferred to a bottle containing 6 liters of inoculum medium and aerated for 24 hours at 28 C. to encourage further growth. The resulting mash is used to inoculate a tank containing the fermentation medium mentioned previously.

The temperature for tank fermentation is 20-35" C., usually 28 C. Aeration is provided by forcing sterile air through or onto the surface of the mash (0.2-2.0, usually 1, liters of air per liter of mesh per minute). Further agitation is provided by a mechanical impeller (120-160 r.p.m.). An anti-foam agent such as 1% octadecanol in lard oil is added as needed. The fermentation time varies from 72 to 140 hours.

Purification procedure After the fermentation is completed, the culture broth containing the antibiotic is filtered at the harvest pH of 6-7 to remove the myceliurn from the broth. Diatoma- 6 ceous earth or any of the conventional filtration aids may be used to assist the filtration which is carried out using standard equipment. Thereafter the antibiotic may be recovered from the filtrate by appropriate extraction procedures.

The antibiotic of this invention may be extracted from the filtrate with solvents such as ethyl acetate, chloroform, n-butanol, etc. at pH 5 to 7 and the extract is concentrated with addition of 0.1 M phosphate buifer pH 6.5.

rThe aqueous concentrate is first extracted with carbon tetrachloride, petroleum ether or hexane to remove impurities, then with chloroform, ethyl acetate or n-butanol which extracts the activity. The chloroform extract is concentrated and may be used for partition chromatography on diatomaceous earth. The developing solvent system may be a mixture of benzene, methanol and phosphate buffer pH 6.5 (20:1:1 by volume). The purple "y2 fraction obtained may be rechromatographed on silicio acid with the solvent system benzene, ethyl acetate, methanol and phosphate buffer pH 6.5 (15:5:121 by volume). The purple eiuent band is concentrated and crystallized from a mixture of ethyl acetate and petroleum ether.

The novel antibiotic of this invention is composed of the elements carbon, hydrogen, nitrogen and oxygen. The antibiotic has the following elemental analysis in percent: carbon, 55.2; hydrogen, 5.9; nitrogen, 16.0 and oxygen, 22.6; O-alkyl, 4.1 as methyl; N-alkyl, 3.7 as methyl; C-alkyl, 3.1 methyl; active hydrogen, 0.99. These analytical values establish the empirical formula:

The molecular weight by X-ray diffraction is 351i10. The antibiotic has a melting point of 202-204 C. The product has an optical rotation of []D25=-242 (t) (c., 0.045% in absolute methanol). A methanolic solution of the product has maxima in the ultraviolet and visible regions at An infrared absorption spectrum of the antibiotic was prepared in a standard manner by mixing with crystals of KBr and pressing into a disc. The compound exhibits characteristics absorption in the infrared region of the spectrum at the following wave lengths expressed in microns: 2.90, 3.00, 3.26, 3.39, 5.84, 6.02, 6.18, 6.32, 6.74, 6.88, 7.03, 7.10, 7.24, 7.37, 7.49, 7.94, 8.16, 8.53, 8.72, 9.01, 9.16, 9.28, 9.49, 9.63, 9.81, 10.15, 10.40, 10.77, 11.12, 11.69, 12.32, 12.69, 13.07, 13.58, 13.84, 14.33, and 14.65. The infrared curve is shown in the accompanying drawing.

The antibiotic shows the following Rf values in the solvent systems indicated below. The organism used for the bioautography was B. subtilis.

Rf value of y2: Solvent system 0.1 Benzene, methanol, water (111:2 by volume).

0.6 Benzene, isoayml alcohol,

water (4:1:2 by volurne).

0.3 1,2-dichloroethane, carbon tetrachloride, acetic acid, Water (4:4;1:2 by volume).

7 TABLE 3 Zones of inhibition Organism: in mm.

Bacillus cereus 3.0 Klebsiella pneumoniae (Friedlanders) 6.0 Alcaligenes ATCC 10153 H ormodendrum cladosporoides 0 Bacillus subtilis Led. #17 10.9 *14.9

Bacillus subtilis Led. #18 4.0 Mycobacterium #607 19.0 f" l5.0

Staphylococcus aureus Led. #27 6.0 Klebsiella pneumoniae sl. Escherichia coli Led. #29 0 Escherichia coli Led. 30 0 Streptococcus pyogenes NY-5 12.0 Staphylococcus aureus ATCC 6538 7.5 Corynebacterium xerose NRRL Bl397 11.3 Erwinia amylovora 6.0

Salmonella gallinarum #605 0 Pasteurella multocida 5.0 Staphylococcus aureus Smith Strain 10.1 Klebsiella pneumoniae A Strain AD sl. Candida albicans Strain CA300 0 *pH 6 agar.

The new antibiotic is highly active in vitro against selected strains of gram-negative and gram-positive bacteria and mammalian cells.

In vivo the new antibiotic has been found to be eiiective against mammary adenocarcinoma 72j in mice.

In vivo it is as effective as some of the other commonly used antibiotics such as tetracycline in protecting mice infected with Staphylococcus Smith. The novel antibiotic is also effective against Leptospira pomona in chick embryos. The novel antibiotic is also effective against pleuro-pneumonia-like organisms`referred` to usually as PPLO and known to be one of the etiological agents which causes the very serious chronic respiratory disease of poultry.

The novel antibiotic of this invention is also particularly useful in protecting chickens against infections with Pasteurella multocida. This organism is the etiological agent responsible for fowl cholera.

In tests involving the infection of one-week old Hallcross sex-linked cockerels by intracoelomic injection of 0.5 milliliter of a 1/1000 dilution of an 18 hour culture of Pasteurella multocida, followed by intracoelomic injection a half-hour later of the novel antibiotic at a dosage of 25 mg./kg., 19 out of 20 animals survived of the total number of animals treated. With the infected controls, however, none of the animals survived.

The new antibiotic has not as yet been demonstrated to be useful in human therapy.

The invention will be described in greater detail in conjunction with the following specific examples.

Example 1.-Fermentation of culture AB-929 (ATCC No. 13,495)

Four 500 milliliter flasks of inoculum were prepared as described under inoculum preparation. These inoculum asks were used to seed two liter bottles containing 12 liters each of fermentation medium. The fermentation medium was that previously described except it did not contain ammonium sulfate. The medium before inoculation was sterilized by procedures well known by those skilled in the art. The fermentation conditions were as follows:

Agitation Impeller speed 400 r.p.m. Aeration 0.03 c.f.m.

Temperature 26 C. to 28 C.

The fermentation was allowed to proceed for 46.5 hours at which time the bottles were harvested and the broth assayed.

CFI

Example 2.-Tank fermentation of 1B-929 Two 500 milliliter flasks containing milliliters each of medium and one 9 liter bottle containing 6 liters of medium were inoculated and prepared as described previously under inoculum preparations. This inoculum was used to seed a 200 gallon tank containing 500 liters of fermentation medium. The tank medium was sterilized for 60 minutes at 120 C. before inoculation.

The fermentation conditions were as follows:

Temperature 28 C.

Aeration 1.0 liter of air/ liter of mash/ minute. Agitation Impeller speed r.p.m.

Antifoam Hodag oil (an antifoam agent manufactured by Hodag Chemical Corporation, Skokie, Illinois).

The initial pH of the fermentation broth was 7.0, and the fermentation was allowed to continue for 731/2 hours at which time the mash was harvested. The harvest mash, pH 6.4, was bioassayed against B. subtilis and Streptococcus pyogenes.

Example 3.-Isolation of a purified preparation of the antibiotic The mash (1500 liters) was aerated from harvest right up to the moment of filtration in order to prevent the destruction of the activity. The pH of the mash was 6-7. Filtration was carried out With the addition of diatomaceous earth (36 kilograms). The filtrate (1200 liters) was counter-currently extracted in a Luwesta extractor with ethyl acetate (1000 liters) and the extract was concentrated to 300 milliliters. The precipitate which formed was removed by filtration and the filtrate made up to 500 milliliters with ethyl acetate. This solution (250 milliliters) was concentrated to a heavy syrup. Sea sand (600 grams) and diatomaceous earth (120 grams) were intimately mixed with the syrup. The mixture was extracted five times with Water at pH 5 (5 x 700 milliliters). The aqueous phase was adjusted to pH 6.5 and li-poid materials were extracted with hexane (twice with 1/s volume). The aqueous phase was then extracted with ethyl acetate (3 times 1A; volume). The ethyl acetate extract was concentrated almost to dryness in vacuo at 30 C. and extracted with Water. This aqueous extract was lyophilized (16.5 grams) and one-fourth was used as charge for the first partition chromatographic column.

Example efr-Isolation of a purified preparation of the antibiotic modified procedure The aerated mash was filtered at harvest pH 6-7 and the mycelium discarded. The beer was extracted with ethyl acetate; phosphate buffer of pH 6.5 was added to the extract. Ethyl acetate was removed, leaving an `aqueous phase. Lipoid material was extracted with petroleum ether. The aqueous phase was extracted with chloroform, buffer `of pH 6.5 was added to the extract which was then evaporated to dryness and used as charge for a Celite column.

Example 5 .-Isolation in crystalline form and characterization of the antibiotic Material (5.5 grams) prepared by the method of Example 3 was chromatographed on a Celite column (900 grams) by development with a system containing benzene, methanol and phosphate buffer pH 6.5 (120:1:1 by volume). The purple-blue band obtained was evaporated to dryness at 35 C. under vacuum. The residue was dissolved in lower phase (21 milliliters) of the system benzene, ethyl acetate, methanol, aqueous phosphate buffer pH 6 (812:1:1 by volume) and thoroughly mixed with silicio acid (30 grams). This charge was fractionated on a silicic acid `column grams) with the upper phase of the above solvent system. The purple band obtained was evaporated to dryness and dissolved in ethyl acetate.

9 Upon addition of Skellysolve B dark blue needles (480 milligrams) were obtained. Upon recrystallization from ethyl acetate-petroleum ether, the yield of crystalline antibiotic was 410 milligrams. The chemical analysis of this product and its other chemical, physical and biological graphic Rf numbers of 0.1 in the system benzene, methanol, water (1:1:2 by volume), of 0.6 in the system benzene, isoamyl alcohol, water (4:2:1 by volume) and 0.3 in the system 1,2-dichloroethane, carbon tetrachloride, acetic acid, Water (4:4: 1:2 by volume).

properties have already been described. 5 2. The antibiotic porromycin as set forth in claim 1 in its essentially pure form. Example 6 3. A substance elfective in inhibiting the gnowth of The PrQCedUIe f EXamPleS 2 and 5 Were relleated SIUE lgram-positive and gram-negative bacteria, said substance the 011831115111 ATCC N0. 13,53@ (AA-849) The C0111- 0 crystallizing in dark purple needles, melting at 1202-204" pound Obtained Was the Same aS H1 EXample 5- l C., which contains the elements carbon, hydrogen, ni-

Example 7 trogen, and oxygen in substantially the following percentages by weight:

The procedure Iof Example 1 was followed usmg the Carbon 55 2 organism ATCC No. 13,539 (AB-286). The product l H dr I; 5'9 was subjected to chromatographic examination and was Nr ege 16'0 the same as produced in Example 5. O1 Ogen 22'6 The antibiotic of the present invention is distinguishable Xygen from other red-purple-blue antibiotics shown in the folhaving an optical rotation [ot]D:-{242 (i100) (c., lowing table: 0.045 percent in methanol), a methanolic solution of said TABLE 4 Antibiotic me UV max. M.P., (3. M.W Indicator Remarks .Actinoflocim- 230, 270 .Actinomycins Chromopeptides. Actinorhodin 285, 52 531, 571 Contains no N. Bostrycoidin 251, 320 Insol. in H20. Coelicolorin Hdirgisol. below pH 8, green at Cyanomycim--- 240, 278, 384 (0.1 N HC1) 0551112151202. Isorhodomycin 240, 310, 551, 563, 610 Czn-21Hn-31OsN.HCl. Iodim'n Insol. in alcohol and H2O. An

acid produced by bacterium. Javanicin 303, 505 in ethanol No N; produced by fungus. Lactaroviolin 242, 314, 398, 527 in EOH Do. Litmocidin Insel. in H2O. Inact. against Staph. in mice. Mycorhodin 25S, 420, 471 N 215, 818, 530 Separated by paper chromatography; IR E1% 1 cm. 22o, 320, 55o Do. Mitomycin C 216, 360, 560 D0. Pluramycin B. Insol. H2O. Prodigiosin- 225,B2:8S,4)337, 471, 539 (EtOH Insol. in H2O; CgoHgONa.

p 7. :Pyocyanine InSOl. 1D benzene; CuHwONg.

Produced by bacterium. Ractinomycin 245, 4410-450 None 692 CaaHoNaOu. Rhodocfiin 515 Difterentiated by distribution coeiicients. Rhodomycetin 235, 520, 540, 580 300 Yes Inactig'e) to Staph. in mice; insol.

1n 2 A: 498 532, 566 A: 193 Separated by paper chromatog- Rhodomycms {Bz 'atcl 420 Yes raphy. nubid'm 520 Nag-)f No N, stam@ 1n and.

Rubromycin Insol. in H2O; No N. Spinninsn No opt. rot.; no N; produced by fungus. Insol. cold H2O.

We claim: substance having maxima in the ultraviolet and visible said substance when suspended in a potassium bromide pellet exhibiting characteristic absorption in the infrared region of the spectrum at the following wavelengths expressed in microns: 2.90, 3.00, 3.26, 3.39, 5.84, 6.02, 6.18, 6.32, 6.74, 6.88, 7.03, 7.10, 7.24, 7.37, 7.49, 7.94, 8.16, 8.53, 8.72, 9.01, 9.16, 9.28, 9.49, 9.63, 9.81, 10.15, 10.40, 10.77, 11.12, 11.69, 12.32, 112.69, 13.07, 13.58, 13.84, 14.33 and 14.65, and said substance having chromatoregions of the spectrum `at 216 ma, 358 mp. and 550 ma, said substance exhibiting characteristic absorption in the infrared region of the spectrum as shown in FIG. 1 and having chromatographic Rf numbers of 0.1 in the system benzene, methanol, Water (111:2 by volume), 0.6 in the system benzene, isoamyl alcohol, Water (4:2:1 by volume) and 0.3 in the system 1,2-dichloroethane, carbon tetrachloride, acetic acid, water (4:4:1:2 by volume), said substance being in crystalline form.

4. A process for the production of portiromycin which comprises cultivating a porromycin-producing organism selected from the group consisting of Streptomyces verticl'llatus ATCC No. 13,495, Streptomyces verfcillatus ATCC No. 13,538 and Streptomyces vertcz'latus ATCC No. 13,539, in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions until substantial anti-bacterial activity is imparted to said medium.

No references cited.

JULIAN S. LEVITT, Primary Examiner.

MORRIS 0. WOLK, IRVING MARCUS, Examiners'. 

1. A SUBSTANCE EFFECTIVE IN INHIBITING THE GROWTH OF GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA, SAID SUBSTANCE CRYSTALLIZING IN DARK PURPLE NEEDLES, MELTING AT 202-204* C., HAVING THE FOLLOWING COMPOSITION IN PERCENT ON ELEMENTAL ANALYSIS: CARBON, 55.5, HYDROGEN, 5.8, NITROGEN, 16.0, OXYGEN, 22.6, THE SAID ANALYTICAL VALUES ESTABLISHING THE EMPIRICAL FORMULA C16H20N4O5, SAID SUBSTANCE HAVING A MOLECULAR WEIGHT OF 351$10, HAVING AN OPTICAL ROTATION (A)D25*+242* ($100*) (C., 0.045 PERCENT IN METHANOL), A METHANOLIC SOLUTION OF SAID SUBSTANCE HAVING MAXIMA IN THE ULTRAVIOLET AND VISIBLE REGIONS OF THE SPECTRUN AT 216MU (E=665), 358MU (E=638), 550MU (E=6.5) SAID SUBSTANCE WHEN SUSPENDED IN A POTASSIUM BROMIDE PELLET EXHIBITING CHARACTERTISIC ABSORPTION IN THE INFRARED REGION OF THE SPECTRUM AT THE FOLLOWING WAVELENGTHS EXPRESSED IN MICRONS: 2.90, 3.00, 3.26, 3.39, 5.84, 6.02, 6.18, 6.32, 6.74, 6.88, 7.03, 7.10, 7.24, 7.37, 7.49, 7.94, 8.16, 8.53, 8.72, 9.01, 9.16, 9.28, 9.49, 9.63, 9.81, 10.15, 10.40, 10.77, 11.12, 11.69, 12.32, 12.69, 13.07, 13.58, 13.84, 14.33 AND 14.65, AND SAID SUBSTANCE HAVING CHROMATOGRAPHIC RF NUMBERS OF 0.1 IN THE SYSTEM BENZENE, METHANOL, WATER (1:1:2 BY VOLUME), OF 0.6 IN THE SYSTEM BENZENE, ISOAMYL ALCOHOL, WATER (4:2:1 BY VOLUME) AND 0.3 IN THE SYSTEM 1,2-DICHLOROETHANE, CARBON TETRACHLORIDE, ACETIC ACID, WATER (4:4:1:2 BY VOLUME). 